Characterization of the complete chloroplast genome of ‘Quanhong poplar’ (Populus deltoides W. Bartram ex Humphry Marshall, 2011)

Abstract The color of the leaves is one of the most important factors for horticultural crops that are considered by breeders, and is also attracting more and more attention from economists and academics. 'Quanhong poplar’ (QHP), a rare, bright reddish-purple color-leaf cultivar that has been widely cultivated in China as a landscape tree, is a very precious color-leaf cultivar. In the present study, a reference-based assembly was performed using whole-genome sequencing data to characterize the chloroplast genome of 'QHP'. The total chloroplast genome size of ‘QHP’ is 156,950 bp, which is divided into two inverted repeat structures of 27,649 bp each, a small single-copy region of 16,563 bp, and a large single-copy region (LSC) of 85,089 bp. From the chloroplast genome, 130 genes have been predicted, including 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. A chloroplast genome containing 36.68% GC content was detected in 'QHP'. Three SNP sites have been developed between 'QHP' and Populus deltoides Zhonglin 2025. Based on the phylogenetic analysis of chloroplast genomes reported for Populus, the chloroplast of 'QHP' is closest to several strains of Populus deltoides.


Introduction
The color-leaf plants are increasingly important in urban beautification, which also greatly contribute to societal and economic developments.The Populus plants, widely used in ecological shelterbelt, agricultural forest shelterbelt, and industrial timber forest, have the characteristics of fast growth, strong adaptability, easy reproduction, and cultivation.Combined with these benefits, color-leaf cultivars in Populus have great potential for development in urban roads and landscaping, such as bright and reddish-purple leaves poplar (Populus deltoides cv.'Quanhong'), red-leaf poplar (Populus deltoides cv.'Zhonghong'), and bright red-leaf poplar (Populus deltoides cv.'Caihong', Populus deltoides cv.'Zhongshancaiyun').Due to its bright and attractive color and high ornamental value, 'Quanhong poplar' (QHP) is a treasure in the red-leaf tree species.Recently, the complete chloroplast genome sequence of Populus deltoides cv.'Caihong' has been characterized (Zhuang et al. 2021).However, the complete chloroplast genome sequence of 'QHP' has not been evaluated.The present study characterizes the chloroplast genome sequence of 'QHP' in order to examine its physiology, molecular mechanisms, and phylogenetical relationships.

Materials and methods
The leaves of 'QHP' (Figure 1) were obtained from Nanjing Botanical Garden, Memorial Sun Yat-sen (E118_83, N32_06), Nanjing, China.The specimen of 'QHP' with voucher number NBG36 was deposited at the Herbarium of Nanjing Botanical Garden, Memorial Sun Yat-sen (http://www.jib.ac.cn/,Weibing Zhuang, weibingzhuangnj@sina.com).No endangered or protected species were involved in the study, and specific permissions were not required for the sample collection.Plant material collection followed local regulations and obtained local authorities' approval.DNA was extracted using the plant DNA isolation reagent (Code: D9194, TaKaRa, Beijing, China) as per the provided instructions.After assessing DNA purity and integrity, qualified DNA underwent library construction and sequencing on the Illumina NovaSeq 6000 system (Illumina Inc., San Diego, CA).A total of 6136.1 Mb raw data were generated, with 3791.4Mb of clean data utilized for chloroplast genome reconstruction.De novo genome assembly and annotation were performed using NOVOPlasty (Dierckxsens et al. 2017) and GeSeq (Tillich et al. 2017), respectively.The chloroplast genome sequence of Populus trichocarpa (NC_009143.1)served as a reference (Tuskan et al. 2006).The sequencing depth coverage was conducted by Geneious (Kearse et al. 2012).The map of chloroplast genome, cis-splicing genes and trans-splicing gene of 'QHP' was drawn by CPGview (http://www.1kmpg.cn/cpgview/,Liu et al. 2023).The SNP calling was performed using SNP-sites with the default options based on the multiple sequence alignments of chloroplast genome sequences (Page et al. 2016).
Multiple sequence alignments encompassing the complete chloroplast genomes of three poplar plants were executed using MAFFT to elucidate sequence disparities.The calculation of nucleotide variability (Pi) was conducted by DnaSP v6 with 600 bp window length and 200 bp step size (Rozas et al. 2017).In order to develop the novel molecular markers between 'QHP' and Populus deltoides Zhonglin 2025, distinctive primers were meticulously designed through Primer Premier 5.
A comprehensive phylogenetic analysis was undertaken using 23 complete chloroplast genomes from the 'Populus' genus, along with three Salix taxa included as outgroups.The sequences were aligned utilizing MAFFT v7.309 (Katoh and Standley 2013).For the purpose of model selection, the GTR-GAMMA (GTR þ G) model was chosen through Modeltest (Posada and Crandall 1998), with the selection criterion being the Bayesian information content (BIC).The maximum-likelihood (ML) method with 1000 bootstraps was employed using MEGA-X software (Kumar et al. 2018).

Results
The complete chloroplast genome of 'QHP' was assembled with a coverage of 3788� (Figure S1).The annotated chloroplast genome was submitted to GenBank under accession number OP115875.Raw sequencing reads were also deposited in the public repository SRA with accession number PRJNA 862560.The chloroplast genome size of 'QHP' measures 156,950 bp, comprising two inverted repeat regions (IRs) each spanning 27,649 bp, a large single-copy (LSC) region of 85,089 bp, and a small single-copy (SSC) region of 16,563 bp (Figure 2).The overall GC content of the 'QHP' chloroplast genome is 36.68%.Analysis revealed 130 genes predicted from this chloroplast genome, consisting of 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes.In addition, the structures of the 12 protein-coding trans-and cis-splicing genes are shown in Figure S2.Comparative analysis of the complete chloroplast genome sequence of 'Populus deltoides' (accession number OP115875) was conducted against other cultivars, such as 'Populus fremontii' (KJ664926), 'Populus trichocarpa' (EF489041), and 'Populus deltoides' (MW165890), utilizing the online genome alignment tool mVISTA (Figure S3).The four genomic sequences exhibit notable similarity, yet substantial sequence variations were observed when comparing 'Populus deltoides' OP115875 with the other cultivars.These variations were primarily concentrated within the LSC and SSC regions (Figure S3).As is typical for most terrestrial plants, the IR regions demonstrated a higher degree of conservation than the LSC and SSC regions.As depicted in Figure 3, all Populus species formed a single large cluster, while the three distinct Salix varieties clustered together in a more compact arrangement.Notably, 'QHP' exhibited a close evolutionary kinship with several kinds of Populus deltoides (Figure 3).
In addition, sliding window analysis was conducted to reveal the highly variable regions in three Populus deltoides chloroplast genomes.The average value of nucleotide diversity (Pi) over the entire chloroplast genome was less than 0.01, indicating the whole cp genome was relatively conserved (Figure S4), which was consistent with the mVISTA result (Figure S3).The SNP calls based on the alignments of chloroplast genome sequences were evaluated (Excel S1).To develop the novel SNP sites between 'QHP' and Populus deltoides Zhonglin 2025, distinctive primers were designed in the regions with Pi > 0.001 (Table S1).The fragments were acquired with the expected size according to the agarose gel electrophoresis results (Figure S5).There was one base difference in the sequence of three different genes between 'QHP' and Populus deltoides Zhonglin 2025 (Table S1), which indicated that these three pairs of primers can be used as potential molecular markers.

Discussion and conclusions
This is further underscored by the meticulous comparison of the 'QHP' plastome with preexisting published datasets, revealing a substantial degree of gene synteny shared with all publicly accessible data on different species of Populus spp.(Wang et al. 2016;Han et al. 2017;Zhuang et al. 2020Zhuang et al. , 2021)).The comprehensive chloroplast genome sequence of 'QHP' holds significant value as a resource for DNA super-barcoding and the analysis of the phylogenetic origins within the genus 'Populus'.

Figure 1 .
Figure 1.Morphology of QHP.The photo was taken by Weibing Zhuang at Nanjing Botanical Garden, Memorial Sun Yat-sen (E118_83, N32_06), Nanjing, China, on 24 June 2020, without any copyright issues.Leaves and new shoots of QHP were dark purple from bud flush in spring to late June, then showing a medium shade of purple from July to September, and turning into bright red in October.The bright and reddish-purple leaves among 'QHP' are the main characters distinct from other poplar cultivars.

Figure 2 .
Figure 2. The chloroplast genome map of QHP.Genes shown outside the circle are transcribed clockwise, and genes inside are transcribed counter-clock-wise.Genes belonging to different functional groups are color-coded.The darker grey in the inner corresponds to the GC content and the lighter grey to the AT content.